Method of treating disturbances of iron distribution in inflammatory intestinal diseases

ABSTRACT

The present invention relates to the use of erythropoietin for the treatment of disturbances of iron distribution in chronic inflammatory intestinal diseases.

PRIORITY TO RELATED APPLICATIONS

This application claims foreign priority to EP 03104832.5, filed Dec.19, 2003.

FIELD OF THE INVENTION

The present invention relates to a new use of erythropoietin in thetreatment of disturbances of iron distribution in patient's sufferingfrom chronic inflammatory intestinal diseases.

BACKGROUND OF THE INVENTION

Various diseases are known in which the metabolism of iron is notnormal. For example, in an anemia not enough blood can be formed due toan overall lack of iron in the body. Another metabolic conditionrelating to iron is hemochromatosis, in which the overall concentrationof iron in the body is higher than normal, which can lead to variousdeleterious health conditions, including the destruction of organs.

Disturbances of iron distribution differ from the above-describedmetabolic disorders (anemia and hemochromatosis) in that the overallconcentration of iron in the body is normal. However, the ironconcentrations are misdistributed throughout the body. Thus excess ironaccumulates in various organs and can lead to damage, even destruction,of these organs. On the other hand, less than normal quantities of ironare available for the formation of blood, leading to secondary effectswhich are comparable to those related to anemia.

Until now it was not known that patients suffering from chronicinflammatory intestinal diseases have a high probability being affectedby disturbances of iron distribution. Disturbances of iron distributioncan be diagnosed by various parameters which are commonly used in thediagnosis of the iron status. Based on measurements of ferritin andsoluble transferrin receptor it is possible to assess whether theoverall concentration of iron in a patient suffering from chronicinflammatory intestinal diseases is normal. If this is the case, then alowered concentration of Hemoglobin in reticulocytes is an indicator fordisturbances of iron distribution. Another indicator is acontinuously/prolonged elevated concentration of C-reactive protein(CRP) in patients suffering from chronic inflammatory intestinaldiseases and exhibiting a normal overall iron concentration. A methodfor diagnosing disturbances of iron distribution has been described byP. Lehmann, M. Volkmann, J. Lotz, A. Baldauf, R. Roeddiger, posterpresented at the AACC/CSCC, Annual Meeting, Jul. 29-Aug. 2, 2001,Chicago, Ill.

SUMMARY OF THE INVENTION

So far, no treatment has yet been suggested for patients with chronicinflammatory intestinal diseases suffering from disturbances in irondistribution. It is thus an object of the present invention to provide amethod of treatment of disturbances of iron distribution in patientssuffering from chronic inflammatory intestinal diseases therebyminimizing or suppressing the above-mentioned medical problems.

It has surprisingly been found that erythropoietin has a beneficialeffect on disturbances of iron distribution in patients afflicted withchronic inflammatory intestinal diseases. According to the method ofpresent invention, a disturbance of iron distribution in a chronicinflammatory intestinal disease is treated with erythropoietin.

DESCRIPTION OF DRAWINGS

FIG. 1: Primary structure of mature human EPO (165 amino acids) (SEQ IDNO:1).

FIG. 2: Primary structure of human EPO prior to excretion from cells(166 amino acids) (SEQ ID NO:2).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless otherwise indicated the following definitions are set forth toillustrate and define the meaning and scope of the various terms used todescribe the invention herein.

The term “lower-alkyl” as used herein means a linear or branched alkylgroup having from one to six carbon atoms. Examples of lower-alkylgroups include methyl, ethyl and isopropyl, preferably methyl.

The term “lower-alkoxy” as used herein means a group R′—O—, wherein R′is a lower-alkyl as described above.

The term “disturbances of iron distribution in chronic inflammatoryintestinal diseases” refers to a disturbance of iron distribution whichoccurs in patients suffering from chronic inflammatory intestinaldiseases. The disturbance of iron distribution can e.g. be characterizedas described above. Particularly, a disturbance of iron distribution ischaracterized by the following parameters: concentration of solubletransferrin receptor [mg/L] divided by log(concentration of ferritin[μg/L]) is smaller than about 3.5 and simultaneously concentration ofC-reactive protein is above about 5 mg/L.

The term “erythropoietin” or “erythropoietin protein” refers to aprotein with the in vivo biological activity of causing bone marrowcells to increase production of reticulocytes and red blood cells andselected from the group consisting of human erythropoietin and analogswhich are defined below.

The term “pegylated erythropoietin (Peg-EPO or PEG-EPO)” refers to anerythropoietin protein which is covalently linked with one to threepolyethylene derivatives as described below.

In more detail, the present invention relates to the use oferythropoietin in the manufacture of a medicament for the treatment ofdisturbances of iron distribution in chronic inflammatory intestinaldiseases. Examples of chronic inflammatory intestinal diseases treatablepursuant to the method of the present invention are, for example, morbuscrohn, also referred to as crohn's disease, and colitis ulzerosa. In apreferred embodiment, the invention relates to a method as definedabove, wherein the chronic inflammatory intestinal disease is morbuscrohn. In another preferred embodiment, the invention relates to amethod as defined above, wherein the chronic inflammatory intestinaldisease is colitis ulzerosa.

The present invention is especially useful for the preparation ofpharmaceutical compositions comprising erythropoietin aspharmaceutically active ingredient. The term “erythropoietin” or“erythropoietin protein” or “EPO” is as follows: particularly the termsrefer to a glycoprotein, e.g. the human erythropoietin, e.g. having theamino acid sequence set out in (SEQ ID NO: 1) or (SEQ ID NO: 2) or anamino acid sequence substantially homologous thereto, whose biologicalproperties relate to the stimulation of red blood cell production andthe stimulation of the division and differentiation of committederythroid progenitors in the bone marrow. As used herein, these termsinclude such proteins modified deliberately, as for example, by sitedirected mutagenesis or accidentally through mutations. These terms alsoinclude analogs having from 1 to 6 additional sites for glycosylation,analogs having at least one additional amino acid at the carboxyterminal end of the glycoprotein, wherein the additional amino acidincludes at least one glycosylation site, and analogs having an aminoacid sequence which includes a rearrangement of at least one site forglycosylation. These terms include both natural and recombinantlyproduced human erythropoietin. In a preferred embodiment of the presentinvention, the erythropoietin protein is a human erythropoietin havingthe amino acid sequence SEQ ID NO:1.

As set out in detail below, the preparation and purification of EPO arewell known in the art. By erythropoietin is meant the natural orrecombinant protein, preferably human, e.g. epoetin alfa or epoetinbeta, as obtained from any conventional source such as tissues, proteinsynthesis, cell culture with natural or recombinant cells. Any proteinhaving the activity of erythropoietin, such as muteins or otherwisemodified proteins, is encompassed. In a preferred embodiment of thepresent invention, the erythropoietin protein is epoetin alfa or epoetinbeta. Recombinant EPO may be prepared via expression in CHO-, BHK- orHeLa cell lines, by recombinant DNA technology or by endogenous geneactivation. Expression of proteins, including, by endogenous geneactivation, is well known in the art and is disclosed, for example inU.S. Pat. Nos. 5,733,761, 5,641,670, and 5,733,746, 5,994,127,5,733,761, 5,641,670, 6,528,313, 5,981,214 and 5,272,071, the contentsof each of which are incorporated herein by reference. The use asdefined above, wherein the erythropoietin protein is expressed byendogenous gene activation, is preferred. The preferred EPO species forthe preparation of erythropoietin glycoprotein products are human EPOspecies. More preferably, the EPO species is the human EPO having theamino acid sequence set out in SEQ ID NO:1 or SEQ ID NO:2, morepreferably the amino acid sequence SEQ ID NO:1. A preferred embodimentof the present invention therefore relates to the use as describedabove, wherein the erythropoietin protein has the amino acid sequence ofSEQ ID NO:1 or SEQ ID NO:2.

Further, erythropoietin may be a glycoprotein analog having from 1 to 6additional sites for glycosylation. Therefore, the present inventionalso relates to the use as described before, wherein the erythropoietinprotein has the sequence of human erythropoietin modified by theaddition of from 1 to 6 glycosylation sites. Glycosylation of a protein,with one or more oligosaccharide groups, occurs at specific locationsalong a polypeptide backbone and greatly affects the physical propertiesof the protein such as protein stability, secretion, subcellularlocalization, and biological activity. Glycosylation is usually of twotypes. O-linked oligosaccharides are attached to serine or threonineresidues and N-linked oligosaccharides are attached to asparagineresidues. One type of oligosaccharide found on both N-linked andO-linked oligosaccharides is N-acetylneuraminic acid (sialic acid),which is a family of amino sugars containing 9 or more carbon atoms.Sialic acid is usually the terminal residue on both N-linked andO-linked oligosaccharides and, because it bears a negative charge,confers acidic properties to the glycoprotein. Human erythropoietin,having 165 amino acids, contains three N-linked and one O-linkedoligosaccharide chains which comprise about 40% of the total molecularweight of the glycoprotein. N-linked glycosylation occurs at asparagineresidues located at positions 24, 38, and 83 and O-linked glycosylationoccurs at a serine residue located at position 126. The oligosaccharidechains are modified with terminal sialic acid residues. Enzymaticremoval of all sialic acid residues from the glycosylated erythropoietinresults in loss of in vivo activity but not in vitro activity becausesialylation of erythropoietin prevents its binding, and subsequentclearance, by hepatic binding protein.

The term “erythropoietin” includes analogs of human erythropoietin withone or more changes in the amino acid sequence of human erythropoietinwhich result in an increase in the number of sites for sialic acidattachment. These glycoprotein analogs may be generated by site-directedmutagenesis having additions, deletions, or substitutions of amino acidresidues that increase or alter sites that are available forglycosylation. Glycoprotein analogs having levels of sialic acid greaterthan those found in human erythropoietin are generated by addingglycosylation sites which do not perturb the secondary or tertiaryconformation required for biological activity. The glycoproteins of thepresent invention also include analogs having increased levels ofcarbohydrate attachment at a glycosylation site which usually involvethe substitution of one or more amino acids in close proximity to anN-linked or O-linked site. The glycoproteins of the present inventionalso include analogs having one or more amino acids extending from thecarboxy terminal end of erythropoietin and providing at least oneadditional carbohydrate site. The erythropoietin proteins of the presentcomposition also include analogs having an amino acid sequence whichincludes a rearrangement of at least one site for glycosylation. Such arearrangement of glycosylation site involves the deletion of one or moreglycosylation sites in human erythropoietin and the addition of one ormore non-naturally occurring glycosylation sites. Increasing the numberof carbohydrate chains on erythropoietin, and therefore the number ofsialic acids per erythropoietin molecules may confer advantageousproperties such as increased solubility, greater resistance toproteolysis, reduced immunogenicity, increased serum half-life, andincreased biological activity. Erythropoietin analogs with additionalglycosylation sites are disclosed in more detail in European PatentApplication 640 619, to Elliot published Mar. 1, 1995.

In a preferred embodiment, the pharmaceutical composition of the presentinvention comprises erythropoietin proteins with an amino acid sequencewhich includes at least one additional site for glycosylation such as,but not limited to, erythropoietins comprising the sequence of humanerythropoietin modified by a modification selected from the following:

Asn³⁰Thr³²; Asn⁵¹Thr⁵³, Asn⁵⁷Thr⁵⁹; Asn⁶⁹; Asn⁶⁹Thr⁷¹; Ser⁶⁸Asn⁶⁹Thr⁷¹;Val⁸⁷Asn⁸⁸Thr⁹⁰; Ser⁸⁷Asn⁸⁸Thr⁹⁰; Ser⁸⁷Asn⁸⁸Gly⁸⁹Thr⁹⁰;; (SEQ ID NO:3)Ser⁸⁷Asn⁸⁸Thr⁹⁰Thr⁹²; Ser⁸⁷Asn⁸⁸Thr⁹⁰Ala¹⁶²; Asn⁶⁹Thr⁷¹Ser⁸⁷Asn⁸⁸Thr⁹⁰;Asn³⁰Thr³²Val⁸⁷Asn⁸⁸Thr⁹⁰; Asn⁸⁹Ile⁹⁰Thr⁹¹; Ser⁸⁷Asn⁸⁹Ile⁹⁰Thr⁹¹;Asn¹³⁶Thr¹³⁸; Asn¹³⁸Thr¹⁴⁰; Thr¹²⁵; and Pro¹²⁴Thr¹²⁵.

The notation used herein for modification of amino acid sequence meansthat the position(s) of the corresponding unmodified protein (e.g. hEPOof SEQ ID NO:1 or SEQ ID NO:2) indicated by the superscripted number(s)is changed to the amino acid(s) that immediately precede the respectivesuperscripted number(s).

The erythropoietin protein may also be an analog having at least oneadditional amino acid at the carboxy terminal end of the glycoprotein,wherein the additional amino acid includes at least one glycosylationsite. The additional amino acid may comprise a peptide fragment derivedfrom the carboxy terminal end of human chorionic gonadotropin.Preferably, the glycoprotein is an analog selected from the groupconsisting of (a) human erythropoietin having the amino acid sequence,Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg Leu ProGly Pro Ser Asp Thr Pro Ile Leu Pro Gln (SEQ ID NO: 4), extending fromthe carboxy terminus; (b) the analog in (a) further comprising Ser⁸⁷Asn⁸⁸ Thr⁹⁰ EPO; and (c) the analog in (a) further comprising Asn³⁰Thr³² Val⁸⁷ Asn⁸⁸ Thr⁹⁰ EPO.

The erythropoietin protein may also be an analog having an amino acidsequence which includes a rearrangement of at least one site forglycosylation. The rearrangement may comprise a deletion of any of theN-linked carbohydrate sites in human erythropoietin and an addition ofan N-linked carbohydrate site at position 88 of the amino acid sequenceof human erythropoietin. Preferably, the glycoprotein is an analogselected from the group consisting of Gln²⁴ Ser87 Asn88 Thr⁹⁰ EPO; Gln³⁸Ser87 Asn88 Thr⁹⁰ EPO; and Gln⁸³ Ser⁸⁷ Asn⁸⁸ Thr⁹⁰ EPO. A further analogis darbepoetin alfa. A preferred erythropoietin protein in the usedescribed before is darbepoietin alfa.

More particularly, the erythropoietin protein of the presentpharmaceutical composition as described above may also include pegylatedderivatives thereof. Pegylated derivatives of erythropoietin and theirpreparation are known in the art and described for example in WO01/02017, EP-A-1064951, EP-A-539,167, EP-A-605,963, WO 93/25212, WO94/20069, WO 95/11924, U.S. Pat. No. 5,56, EP-A-584,876, WO 92/16555, WO94/28024, WO 97/04796, U.S. Pat. Nos. 5,359,030 and 5,681,811,4,179,337, Japanese Patent, WO 98/32466, U.S. Pat. Nos. 5,324,650 and6,583,272 B1. In a preferred embodiment of the invention, theerythropoietin protein used in the described method is pegylated.

Accordingly, the present invention also refers to the use as describedabove, wherein the erythropoietin protein is a conjugate, said conjugatecomprising an erythropoietin protein as described above having at leastone free amino group and having the in vivo biological activity ofcausing bone marrow cells to increase production of reticulocytes andred blood cells and selected from the group consisting of humanerythropoietin and analogs thereof which have a sequence of humanerythropoietin modified by the addition of from 1 to 6 glycosylationsites or a rearrangement of at least one glycosylation site; saiderythropoietin being covalently linked to “n” poly(ethylene glycol)groups of the formula —CO—(CH₂)_(x)—(OCH₂CH₂)_(m)—OR with the —CO (i.e.carbonyl) of each poly(ethylene glycol) group forming an amide bond withone of said amino groups; wherein R is lower-alkyl; x is 2 or 3; m isfrom about 450 to about 900; n is from 1 to 3; and n and m are chosen sothat the molecular weight of the conjugate minus the erythropoietinprotein is from 20 kilodaltons to 100 kilodaltons. This inventionfurther provides pharmaceutical compositions containing conjugatesdescribed herein in which the percentage of conjugates in which n is 1is at least ninety percent, preferably at least ninety-two percent, orepreferably ninety-sex percent of all conjugates of the composition.

More specifically the above conjugates may be represented by formula (I)P-[NHCO—(CH₂)_(x)—(OCH₂CH₂)_(m)—OR]_(n)  (I)wherein P is the residue of an erythropoietin protein as describedherein, (i.e. without the free amino group or amino groups which form anamide linkage with the carbonyl shown in Formula I), having the in vivobiological activity of causing bone marrow cells to increase productionof reticulocytes and red blood cells; and wherein R is lower-alkyl; x is2 or 3; m is from about 450 to about 900; n is from 1 to 3; and n and mare chosen so that the molecular weight of the conjugate minus theerythropoietin glycoprotein is from 20 kilodaltons to 100 kilodaltons.In accordance with this invention, R is any lower-alkyl. Conjugates inwhich R is methyl are preferred.

The symbol “m” represents the number of ethylene oxide residues(OCH₂CH₂) in the poly(ethylene oxide) group. A single PEG (polyethyleneglycol) subunit of ethylene oxide has a molecular weight of about 44daltons. Thus, the molecular weight of the conjugate (excluding themolecular weight of the EPO) depends on the number “m”. In theconjugates of this invention “m” is from about 450 to about 900(corresponding to a molecular weight of about 20 kDa to about 40 kDa),preferably from about 650 to about 750 (corresponding to a molecularweight of about 30 kDa). The number m is selected such that theresulting conjugate of this invention has a physiological activitycomparable to unmodified EPO, which activity may represent the same as,more than, or a fraction of the corresponding activity of unmodifiedEPO. A molecular weight of “about” a certain number means that it iswithin a reasonable range of that number as determined by conventionalanalytical techniques. The number “m” is selected so that the molecularweight of each poly(ethylene glycol) group covalently linked to theerythropoietin glycoprotein is from about 2 kDa to about 40 kDa, and ispreferably about 30 kDa.

In the conjugates of this invention, the number “n is the number ofpoly(ethylene glycol) groups covalently bound to free amino groups(including ε-amino groups of a lysine amino acid and/or theamino-terminal amino group) of an erythropoietin protein via amidelinkage(s). A conjugate of this invention may have one, two, or threePEG groups per molecule of EPO. “n” is an integer ranging from 1 to 3,preferably “n” is 1 or 2, and more preferably “n” is 1. A preferredconjugate of the conjugates described above comprises compounds whereinx is 2, m is 650 to 750, n is 1 and R is methyl.

The compound of formula (I) can be prepared from the known polymericmaterial:

in which R and m are as described above, by condensing the compound ofFormula II with the erythropoietin glycoprotein. Compounds of formula(II) in which x is 3 are alpha-lower-alkoxy, butyric acid succinimidylesters of poly(ethylene glycol) (lower-alkoxy-PEG-SBA). Compounds offormula (II) in which x is 2 are alpha-lower-alkoxy, propionic acidsuccinimidyl esters of poly(ethylene glycol) (lower-alkoxy-PEG-SPA). Anyconventional method of reacting an activated ester with an amine to forman amide can be utilized. In the reaction described above, theexemplified succinimidyl ester is a leaving group causing the amideformation. The use of succinimidyl esters such as the compounds offormula II to produce conjugates with proteins are disclosed in U.S.Pat. No. 5,672,662, issued Sep. 30, 1997 (Harris, et al.).

Human EPO contains nine free amino groups, the amino-terminal aminogroup plus the ε-amino groups of 8 lysine residues. When the pegylationreagent was combined with a SBA compound of Formula II, it has beenfound that at pH 7.5, a protein:PEG ratio of 1:3, and a reactiontemperature of from 20-25° C., a mixture of mono-, di-, and traceamounts of the tri-pegylated species were produced. When the pegylationreagent was a SPA compound of Formula II, at similar conditions exceptthat the protein:PEG ratio was 1:2, primarily the mono-pegylated speciesis produced. The pegylated EPO can be administered as a mixture, or asthe cation exchange chromatography separated different pegylatedspecies. By manipulating the reaction conditions (e.g., ratio ofreagents, pH, temperature, protein concentration, time of reactionetc.), the relative amounts of the different pegylated species can bevaried.

A further preferred embodiment of the present invention relates to theuse as defined above, wherein the erythropoietin protein is a conjugate,said conjugate comprising an erythropoietin protein as defined abovehaving at least one free amino group and having the in vivo biologicalactivity of causing bone marrow cells to increase production ofreticulocytes and red blood cells and selected from the group consistingof human erythropoietin protein and analogs thereof which have theprimary structure of human erythropoietin protein modified by theaddition of from 1 to 6 glycosylation sites; said erythropoietin proteinbeing covalently linked to from one to three lower-alkoxy poly(ethyleneglycol) groups, each poly(ethylene glycol) group being covalently linkedto the erythropoietin protein via a linker of the formula —C(O)—X—S—Y—with the C(O) of the linker forming an amide bond with one of said aminogroups, X is —(CH₂)_(k)— or —CH₂(O—CH₂—CH₂)_(k)—, k is from 1 to 10, Yis

the average molecular weight of each poly(ethylene glycol) moiety isfrom about 20 kilodaltons to about 40 kilodaltons, and the molecularweight of the conjugate is from about 51 kilodaltons to about 175kilodaltons.

This erythropoietin species may also be represented by formula (III)P-[NH—CO—X—S—Y—(OCH₂CH₂)_(m)—OR]_(n)  (III)wherein R may be any lower-alkyl. A preferred lower-alkyl is methyl. Xmay be —(CH₂)_(k)— or —CH₂(O—CH₂—CH₂)_(k)—, wherein k is from 1 to about10. Preferably, k is from 1 to about 4, more preferably, k is 1 or 2.Most preferably, X is —(CH₂).

In formula (III), the number m is selected such that the resultingconjugate of formula (III) has a physiological activity comparable tounmodified EPO, which activity may represent the same as, more than, ora fraction of the corresponding activity of unmodified EPO. m representsthe number of ethylene oxide residues in the PEG unit. A single PEGsubunit of —(OCH₂CH₂)— has a molecular weight of about 44 daltons. Thus,the molecular weight of the conjugate (excluding the molecular weight ofthe EPO) depends on the number m. A molecular weight of “about” acertain number means that it is within a reasonable range of that numberas determined by conventional analytical techniques. m is an integerranging from about 450 to about 900 (corresponding to a molecular weightof from 20 to 40 kDa), preferably m is from about 550 to about 800(about 24 to 35 kDa), and most preferably m is from about 650 to about700 (about 29 to about 31 kDa).

In formula (III), the number n is the number of ε-amino groups of alysine amino acid in an erythropoietin protein covalently bound to a PEGunit via an amide linkage. A conjugate of this invention may have one,two, or three PEG units per molecule of EPO. n is an integer rangingfrom 1 to 3, preferably n is 1 or 2, and more preferably n is 1.

Preferred erythropoietin proteins of formula (III) are represented bythe formulae:

In the most preferred embodiment of the present invention, anerythropoietin conjugate is represented by the formula:

wherein in the above formulae n is an integer from 1 to 3; m is aninteger from 450 to 900; R is lower-alkyl; X is —(CH₂)_(k)— or—CH₂(O—CH₂—CH₂)_(k)—, and P is the residue of the erythropoietin proteinwithout the amino group or groups which form an amide linkage with X.

Other preferred erythropoietin glycoprotein products are represented bythe formulae:

More preferred erythropoietin glycoprotein products are represented bythe formula:

These erythropoietin proteins may be prepared by

-   (a) covalently reacting an ε-amino group of a lysine amino acid of    an erythropoietin protein represented by the formula, P-[NH₂]_(n),    with a bi-functional reagent represented by the formula, Z-CO—X—S-Q,    to form an intermediate with an amide linkage represented by the    formula:    P-[NH—CO—X—S-Q]_(n)-    wherein P is an erythropoietin protein less the amino group which    forms an amide linkage; n is an integer ranging from 1 to 3; Z is a    reactive group, e.g. a carboxylic-NHS ester; X is —(CH₂)_(k)— or    —CH₂(O—CH₂—CH₂)_(k)—, wherein k is from 1 to about 10; and Q is a    protecting group, like alkanoyl, e.g. acetyl.-   (b) covalently reacting the intermediate with an amide linkage from    step (a) with an activated poly(ethylene glycol) derivative    represented by the formula, W-[OCH₂CH₂]_(m)—OR, to form an    erythropoietin glycoprotein product represented by the formula:

-    wherein W is a sulfhydryl reactive form of Y; m is an integer    ranging from about 450 to about 900; R is lower-alkyl; and Y is as    defined above.

In this embodiment, the bi-functional reagent is preferablyN-succinimidyl-S-acetylthiopropionate orN-succinimidyl-S-acetylthioacetate, Z is preferablyN-hydroxy-succinimide, and the activated poly(ethylene glycol)derivative W-[OCH₂CH₂]_(m)—OR is preferably selected from the groupconsisting of iodo-acetyl-methoxy-PEG, methoxy-PEG-vinylsulfone, andmethoxy-PEG-maleimide.

In more detail, the erythropoietin proteins of formula (III) may beprepared by covalent linking of thiol groups to EPO (“activation”) andcoupling the resulting activated EPO with a poly(ethylene glycol) (PEG)derivative. The first step for the preparation of pegylated EPOaccording to the present invention comprises covalent linking of thiolgroups via NH₂-groups of EPO. This activation of EPO is performed withbi-functional reagents which carry a protected thiol group and anadditional reactive group, such as active esters (e.g., asuccinimidylester), anhydrides, esters of sulphonic acids, halogenidesof carboxylic acids and sulphonic acids, respectively. The thiol groupis protected by groups known in the art, e.g., acetyl groups. Thesebi-functional reagents are able to react with the ξ-amino groups of thelysine amino acids by forming an amide linkage. The first step of thereaction is set out below:

EPO, n and X are as defined above and Z is a reactive group known in theart, e.g. a N-hydroxy-succinimide (NHS) substituent of the formula

In a preferred embodiment the activation of the ε-amino lysine groups isperformed by reaction with bi-functional reagents having a succinimidylmoiety. The bi-functional reagents may carry different spacer species,e.g. —(CH₂)_(k)— or —CH₂—(O—CH₂—CH₂—)_(k)— moieties, wherein k is from 1to about 10, preferably from 1 to about 4, and more preferably 1 or 2,and most preferably 1. Examples of these reagents areN-succinimidyl-S-acetylthiopropionate (SATP) andN-succinimidyl-S-acetylthioacetate (SATA)

-   -   Acetylthioalkyl-carboxylic-NHS-ester, like

-   -   2-(Acetylthio)-(ethoxy)k-acetic-acid-NHS-ester        with k as defined above.

The preparation of the bi-functional reagents is known in the art.Precursors of 2-(acetylthio)-(ethoxy)_(k)-acetic-acid-NHS-esters aredescribed in DE-3924705, while the derivatization to the acetylthiocompound is described by March, J., Advanced Organic Chemistry,McGraw-Hill, 1977, 375-376. SATA is commercially available (MolecularProbes, Eugene, Oreg., USA and Pierce, Rockford, Ill.).

The number of thiol groups to be added to an EPO molecule can beselected by adjusting the reaction parameters, i.e., the protein (EPO)concentration and the protein/bi-functional reagent ratio. Preferably,the EPO is activated by covalently linking from 1 to 5 thiol groups perEPO molecule, more preferably from 1.5 to 3 thiol groups per EPOmolecule. These ranges refer to the statistical distribution of thethiol group over the EPO protein population.

The reaction is carried out, for example, in an aqueous buffer solution,pH 6.5-8.0, e.g., in 10 mM potassium phosphate, 50 mM NaCl, pH 7.3. Thebi-functional reagent may be added in DMSO. After completion of thereaction, preferably after 30 minutes, the reaction is stopped byaddition of lysine. Excess bifunctional reagent may be separated bymethods known in the art, e.g., by dialysis or column filtration. Theaverage number of thiol groups added to EPO can be determined byphotometric methods described in, for example, Grasetti, D. R. andMurray, J. F. in J. Appl. Biochem. Biotechnol. 119, 41-49 (1967).

The above reaction is followed by covalent coupling of an activatedpoly(ethylene glycol) (PEG) derivative. Suitable PEG derivatives areactivated PEG molecules with an average molecular weight of from about20 to about 40 kDa, more preferably from about 24 to about 35 kDa, andmost preferably about 30 kDa.

Activated PEG derivatives are known in the art and are described in, forexample, Morpurgo, M. et al. J. Bioconj. Chem. (1996) 7, page 363 ff forPEG-vinylsulfone. Linear chain and branched chain PEG species aresuitable for the preparation of the compounds of Formula 1. Examples ofreactive PEG reagents are iodo-acetyl-methoxy-PEG andmethoxy-PEG-vinylsulfone:

The use of these iodo-activated substances is known in the art anddescribed e.g. by Hermanson, G. T. in Bioconjugate Techniques, AcademicPress, San Diego (1996) p. 147-148.

Most preferably, the PEG species are activated by maleimide using(lower-alkoxy-PEG-maleimide), such as methoxy-PEG-maleimide (MW 30000;Shearwater Polymers, Inc.). The structure of lower-alkoxy-PEG-maleimideis as follows:

with R and m are as defined above, preferably

The coupling reaction with lower-alkoxy-PEG-maleimide takes place afterin situ cleavage of the thiol protecting group in an aqueous buffersolution, e.g. 10 mM potassium phosphate, 50 mM NaCi, 2 mM EDTA, pH 6.2.The cleavage of the protecting group may be performed, for example, withhydroxylamine in DMSO at 25° C., pH 6.2 for about 90 minutes. For thePEG modification the molar ratio of activatedEPO/lower-alkoxy-PEG-maleimide should be from about 1:3 to about 1:6,and preferably 1:4. The reaction may be stopped by addition of cysteineand reaction of the remaining thiol (—SH) groups with N-methylmaleimideor other appropriate compounds capable of forming disulfide bonds.Because of the reaction of any remaining active thiol groups with aprotecting group such as N-methylmaleimide or other suitable protectinggroup, the EPO glycoproteins in the conjugates of this invention maycontain such protecting groups. Generally the procedure described hereinwill produce a mixture of molecules having varying numbers of thiolsprotected by different numbers of the protecting group, depending on thenumber of activated thiol groups on the glycoprotein that were notconjugated to PEG-maleimide.

Whereas N-methylmaleimide forms the same type of covalent bond when usedto block the remaining thiol-groups on the pegylated protein, disulfidecompounds will lead in an intermolecular sulfide/disulfide exchangereaction to a disulfide bridged coupling of the blocking reagent.Preferred blocking reagents for that type of blocking reaction areoxidized glutathione (GSSG), cysteine and cystamine. Whereas withcysteine no additional net charge is introduced into the pegylatedprotein, the use of the blocking reagents GSSG or cystamine results inan additional negative or positive charge.

The further purification of the compounds of formula (III), includingthe separation of mono-, di- and tri-pegylated EPO species, may be doneby methods known in the art, e.g., column chromatography.

Pegylated erythropoietin derivatives preferably contain at least ninetypercent mono-PEG conjugates. Usually mono-PEG conjugates oferythropoietin glycoproteins are desirable because they tend to havehigher activity than di-PEG conjugates. The percentage of mono-PEGconjugates as well as the ratio of mono- and di-PEG species can becontrolled by pooling broader fractions around the elution peak todecrease the percentage of mono-PEG or narrower fractions to increasethe percentage of mono-PEG in the composition. About ninety percentmono-PEG conjugates is a good balance of yield and activity. Sometimescompositions in which, for example, at least ninety-two percent or atleast ninety-six percent of the conjugates are mono-PEG species (nequals 1) may be desired. In an embodiment of this invention thepercentage of conjugates where n is 1 is from ninety percent toninety-six percent.

Pharmaceutical compositions comprising pegylated erythropoietin areknown in the art and are described e.g. in International patentapplication WO 01/87329. Compositions may comprise 10 to 10000 μg of anerythropoietin protein per ml as defined above. Preferably, thecompositions comprise 10 to 1000 μg, e.g. 10, 50, 100, 400, 800 or 2500μg per ml. Further, the compositions may comprise 10 μg to 10000 μgerythropoietin protein per ml, 10-200 mmoll sulfate, 10 to 50 mmol/lphosphate, pH 6.0 to 6.5. This composition may also comprise up to 20 mMmethionine, 1-5% of a polyol (w/v), up to 0.1% pluronic F68 (w/v) andoptionally up to 1 mM CaCl₂. An example of this composition comprises 10μg to 10000 μg erythropoietin protein per ml, 40 mmol/l sulfate, 10mmol/l phosphate, 3% mannitol (w/v), 10 mM methionine, 0.01% pluronicF68 (w/v), pH 6.2. In alternative the composition may comprise 10 μg to10000 μg erythropoietin protein per ml, 10 to 100 mmol/l NaCl, 10 to 50mmol/l phosphate pH 6.0 to 7.0, optionally 1-5% (w/v) of a polyol.Further, this composition may comprise up to 20 mM methionine, up to0.1% pluronic F68 (w/v) and optionally 7.5 μmol/l CaCl₂. Specifically,this composition may comprise 10 μg to 10000 μg erythropoietin proteinper ml, 100 mmol/l NaCl, 10 mM methionine, 0.01% pluronic F68 (w/v), and10 mmol/l phosphate, pH 7.0.

The present invention also refers to the use of the above compositioncomprising 10 μg to 10000 μg erythropoietin protein per ml, 10 to 50mmol/l arginine, pH 6 to pH 6.5, 10 to 100 mmol/l sodium sulfate. Inaddition, this composition may comprise up to 20 mM methionine, up to0.1% pluronic F68 (w/v), optionally up to 1 mmol/l CaCl₂ and optionally1-5% (w/v) of a polyol. Specifically, this composition may 10 μg to10000 μg erythropoietin protein per ml, 40 mmol/l arginine, pH 6.2, 30mmol/l sodium sulfate, 3% mannitol (w/v), 10 mM methionine, 0.01%pluronic F68 (w/v) and optionally 1 mmol/l CaCl₂.

The present invention also contemplates use of compositions comprising10 to 10000 μg/ml erythropoietin, preferably 25 to 2500 μg/mlerythropoietin, and

a) 10 mM sodium/potassium phosphate, 100 mM NaCl, pH 7.0 or

b) 10 mM sodium phosphate, 120 mM sodium sulfate, pH 6.2 or

c) 10 mM sodium phosphate, 40 mM sodium sulfate, 3% mannitol (w/v) , pH6.2 or

d) 10 mM sodium phosphate, 40 mM sodium sulfate, 3% mannitol (w/v), 10mM methionine, 0.01% pluronic F68 (w/v), pH 6.2 or

e) 40 mM arginine, 30 mM sodium sulfate, 3% mannitol (w/v), pH 6.2 or

f) 40 mM arginine, 30 mM sodium sulfate, 3% mannitol (w/v), 10 mMmethionine, 0.01% pluronic F68 (w/v), pH 6.2.

In a preferred embodiment, the invention contemplates use ofcompositions comprising an amount erythropoietin protein of 50, 100,400, 800 or 2500 μg/ml. The most preferred compositions comprise either10 mM sodium phosphate, 40 mM sodium sulfate, 3% mannitol (w/v), 10 mMmethionine, 0.01% pluronic F68 (w/v), pH 6.2 or 40 mM arginine, 30 mMsodium sulfate, 3% mannitol (w/v), 10 mM methionine, 0.01% pluronic F68(w/v), pH 6.2. Further details of such compositions are known from WO01/87329.

The invention also relates to a method for treating disturbances of irondistribution in patients afflicted with chronic inflammatory intestinaldiseases comprising administration of an effective amount oferythropoietin protein as defined above. Furthermore, the inventionrelates to a medicament for treating disturbances of iron distributionin chronic inflammatory intestinal diseases characterized in that itcontains an effective amount of erythropoietin protein. Examples ofchronic inflammatory intestinal diseases in which the current method maybe used to treat disturbances of iron distribution include morbus crohn,also referred to as crohn's disease, and colitis ulzerosa, in particularmorbus crohn. Preferred methods and medicaments as described above arethose wherein the erythropoietin protein is as defined above.

In the treatment of disturbances of iron distribution in inflammatoryintestinal diseases EPO can be administered, for example, at a dosage of150 U/kg body weight twice a week. The dosage can be varied according tothe needs of the individual patient and can also be in a range of e.g.100 to 200 U/kg (“therapeutically effective amount” or “effectiveamount”). Depending of the half life time of the used EPO derivative, adose can be administered between e.g. 1 or 3 times per week. Dependingon the needs of an individual patient, a physician might also choose adifferent dosage. As used herein, “U” denotes an international unit ofan EPO, sometimes also designated as “IU”.

The specific activity of EPO or EPO conjugates in accordance with thisinvention can be determined by various assays known in the art. Thebiological activity of the purified EPO proteins of this invention aresuch that administration of the EPO protein by injection to humanpatients results in bone marrow cells increasing production ofreticulocytes and red blood cells compared to non-injected or controlgroups of subjects. The biological activity of the EPO proteins, orfragments thereof, obtained and purified in accordance with thisinvention can be tested by methods according to Annable, et al., Bull.Wld. Hlth. Org. (1972) 47: 99-112 and Pharm. Europa Spec. IssueErythropoietin BRP Bio 1997(2). Another biological assay for determiningthe activity of EPO protein, the normocythaemic mouse assay, isdescribed in the art (e.g. Pharm. Europa Spec. Issue Erythropoietin BRPBio 1997(2), and the monography of erythropoietin of Ph. Eur. BRP.).

The invention will be better understood by reference to the followingexamples which illustrate but do not limit the invention describedherein.

EXAMPLES Example 1

A middle-aged woman with colitis ulzerosa is checked for disturbances ofiron distribution by determination of the following parameters—CRP (Creactive protein), ferritin and soluble transferrin receptor—asdescribed P. Lehmann, M. Volkmann, J. Lotz, A. Baldauf, R. Roeddiger,poster presented at the AACC/CSCC, Annual Meeting, Jul. 29-Aug. 2, 2001,Chicago, Ill. The results show disturbances of iron distribution. Thepatient is treated subcutaneously with 150/U kg Recormon™ (commerciallyavailable erythropoietin protein from Roche) twice a week for a maximumof 12 weeks. Afterwards, determination of the parameters as describedabove shows an improvement of the disorder of iron deficiency.

Example 2

A middle-aged woman with morbus crohn is checked for disturbances ofiron distribution by determination of the following parameters—CRP (Creactive protein), ferritin and soluble transferrin receptor—asdescribed P. Lehmann, M. Volkmann, J. Lotz, A. Baldauf, R. Roeddiger,poster presented at the AACC/CSCC, Annual Meeting, Jul. 29-Aug. 2, 2001,Chicago, Ill. The results show disturbances of iron distribution. Thepatient is treated subcutaneously with 150/U kg Recormon™ (commerciallyavailable erythropoietin protein from Roche) twice a week for a maximumof 12 weeks. Afterwards, determination of the parameters as describedabove shows an improvement of the disorder of iron deficiency.

1. A method of treating disturbances in iron distribution in a patientsuffering from a chronic inflammatory intestinal disease comprisingadministering a; therapeutically effective amount of humanerythropoietin protein having the amino acid sequence of SEQ ID NO:1,without administering iron.
 2. The method of claim 1, wherein thepatient is suffering from morbus crohn.
 3. The method of claim 1,wherein the patient is suffering from colitis ulcerosa.
 4. A method oftreating disturbances in iron distribution in a patient suffering from achronic inflammatory intestinal disease comprising administering atherapeutically effective amount of human erythropoietin protein havingthe amino acid sequence of SEQ ID NO:1 modified by the addition of up tothree glycosylation sites, without administering iron wherein themodification is selected from the group consisting of: Asn³⁰Thr³²;Asn⁵¹Thr⁵³; Asn⁵⁷Thr⁵⁹; Asn⁶⁹; Asn⁶⁹Thr⁷¹; Ser⁶⁸Asn⁶⁹Thr⁷¹;Val⁸⁷Asn⁸⁸Thr⁹⁰; Ser⁸⁷Asn⁸⁸Thr⁹⁰ Ser⁸⁷Asn⁸⁸Gly⁸⁹Thr⁹⁰ (SEQ ID NO:3);Ser⁸⁷Asn⁸⁸Thr⁹⁰Thr⁹² Ser⁸⁷Asn⁸⁸Thr⁹⁰Ala¹⁶² Asn⁶⁹Thr⁷¹Ser⁸⁷Asn⁸⁸Thr⁹⁰Asn³⁰Thr³²Val⁸⁷Asn⁸⁸Thr⁹⁰; Asn⁸⁹Ile⁹⁰Thr⁹¹; Ser⁸⁷Asn⁸⁹Ile⁹⁰Thr⁹¹;Asn¹³⁶Thr¹³⁸; Asn¹³⁸Thr¹⁴⁰; Thr¹²⁵; and Pro¹²⁴-Thr¹²⁵:

and wherein the position(s) of the corresponding unmodifiederythropoietin protein indicated by the superscripted number(s) ischanged to the amino acid(s) that immediately precede the respectivesuperscripted number(s).
 5. A method of treating disturbances in irondistribution in a patient suffering from a chronic inflammatoryintestinal disease comprising administering a therapeutically effectiveamount of human erythropoietin protein, without administering iron,wherein the protein (EPO) is an analog of SEQ ID NO:1, said analog isselected from the group consisting of: (a) human erythropoietin proteinhaving the amino acid sequence, Ser Ser Ser Ser Lys Ala Pro Pro Pro SerLeu Pro Ser Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro lie Leu Pro Gln(SEQ ID NO: 4), extending from the carboxy terminus; (b) the analog in(a) further comprising Ser⁸⁷ Asn⁸⁸ Thr^(9°) EPO; (c) the analog in (a)further comprising Asn^(3°) Thr³² Va187 Asn⁸⁸ Thr^(9°) EPO; (d) theanalog in (a) further comprising Gln²⁴ Ser⁸⁷ Asn⁸⁸ Thr^(9°) EPO; (e) theanalog in (a) further comprising Gln³⁸ Ser⁸⁷ Asn⁸⁸ Thr^(9°) EPO; (f) theanalog in (a) further comprising Gln⁸³ Ser⁸⁷ Asn ⁸⁸ Thr^(9°) EPO and (g)darbepoetin alfa.
 6. The method of claim 1, wherein the erythropoietinprotein is pegylated.
 7. A method of treating disturbances in irondistribution in a patient suffering from a chronic inflammatoryintestinal disease comprising administering a conjugate of humanerythropoietin protein of SEQ ID NO:1 without administering iron,wherein said conjugate comprises the erythropoietin protein of SEQ IDNO:1 having one to three free amino groups covalently linked to npoly(ethylene glycol) groups of the formula—CO—(CH₂)_(x)—(OCH₂CH₂)_(m)—OR with the —CO of each poly(ethyleneglycol) group forming an amide bond with one of said amino groups;wherein R is a lower-alkyl; x is 2 or 3; m is from about 450 to about900; n is from 1to 3; and n and m are chosen so that the molecularweight of the conjugate minus the erythropoietin protein is from 20kilodaltons to 100 kilodaltons.
 8. The method of claim 7, wherein x is2, m is from about 650 to about 750, n is 1 and R is methyl.
 9. Themethod of claim 7 wherein the conjugate has the formulaP-[NHCO—(CH₂)_(x)—(OCH₂CH₂)_(m)—OR]_(n) wherein P is the residue of theerythropoietin without the free amino group that forms the amidelinkage; R is lower alkyl; x is 2or 3; m is from about 450 to about 900;and n is 1; and wherein m and n are selected such that the molecularweight of the conjugate minus the erythropoietin protein is from about20 kd to about 100 kd.
 10. A method of treating disturbances in irondistribution in a patient suffering from a chronic inflammatoryintestinal disease comprising administering a conjugate of theerythropoietin protein of SEQ ID NO:1 without administering iron,wherein said conjugate comprises the erythropoietin protein of SEQ IDNO:1 having one to three free amino, groups covalently linked to fromone to three lower-alkoxy poly(ethylene glycol) groups, eachpoly(ethylene glycol) group being covalently linked to theerythropoietin protein via a linker of the formula —C(O)—X—S—Y— with theC(O) of the linker forming an amide bond with one of said amino groups,X is —(CHz)k—or —CH2(O—CHz—CHz)k—, k is from 1 to 10, Y is

wherein the average molecular weight of each poly(ethylene glycol)moiety is from about 20 kilodaltons to about 40 kilodaltons, and themolecular weight of the conjugate is from about 51 kilodaltons to about175 kilodaltons.
 11. The method of claim 10, wherein the conjugate hasthe formula:

wherein n is 1; m is an integer from about 450 to about 900; R islower-alkyl; X is —(CH₂)_(k)— or —CH₂(O—CH₂—CH₂)_(k)—, k is 1 to 10 andP is the residue of the erythropoietin protein without the n aminogroups which form an amide linkage with X.
 12. The method of claim 1wherein the amount of human erythropoietin protein administered to thepatient is from about 100 U/kg to about 200 U/kg twice per week.
 13. Themethod of claim 1 wherein the amount of human erythropoietin proteinadministered to the patient is about 200 U/kg once every three to fourweeks.
 14. The method of claim 7 wherein said conjugate comprises theerythropoietin protein of SEQ ID NO:1 having one free amino groupcovalently linked to n poly(ethylene glycol) groups of the formula—CO—(CH₂)_(x)—(OCH₂CH₂)_(m)—OR with the —CO of each poly(ethyleneglycol) group forming an amide bond with one of said amino groups;wherein R is a lower-alkyl; x is 2 or 3; m is from about 450 to about900; n is 1; and n and m are chosen so that the molecular weight of theconjugate minus the erythropoietin protein is from 20 kilodaltons to 100kilodaltons.